The opioid peptide precursor, proenkephalin, is expressed by multiple cell types within the testis, including testicular somatic cells and spermatogenic cells. Opioid receptors are also found on testicular cells, indicating that proenkephalin products function as paracrine factors mediating local interactions important for spermatogenesis. In addition, recent findings by the PI have shown that peptides derived from spermatogenic cells are also stored in the acrosome of mature sperm and are released during the acrosome reaction. It is therefore possible that proenkephalin also has an extratesticular role during the fertilization process. In rat and mouse spermatogenic cells, novel proenkephalin mRNAs are present that are generated from a germ cell-specific promoter in a developmentally regulated manner. A transgenic mouse model has been developed using a reporter gene under the control of the rat proenkephalin germ cell promoter. Transgenes are appropriately expressed in mouse spermatogenic cells in a developmentally correct manner. This system thus serves as a useful model to elucidate cell-type and stage-dependent transcriptional regulation during spermatogenesis. The specific aims of this proposal are: (1) to identify cis-acting elements within the proenkephalin germ cell promoter using transgenic mice and in vitro DNA binding a analysis; (2) to characterize the trans-acting factors that interact with regulatory elements within this germ cell promoter; and (3) to clone these germ cell trans-activators. The ultimate goal is to understand how regulation of trans-acting factor activity contributes to the stage-dependent variation in activity of the proenkephalin germ cell promoter.